This invention relates to a Down""s syndrome screening test for pregnant women.
A triple screen of xcex1-fetoprotein, chorionic gonadotropin and unconjugated estrogen in serum has been suggested for the prenatal diagnosis of Down""s syndrome (Bennett, J. C., and Plum, F., Cecil""s Textbook of Medicine, W. B. Saunders, Philadelphia, 1996, p. 165). However, it allows detection of only 60 to 65% of fetuses with the genetic disorder and gives 5% false positive results. It is also limited to the second trimester of pregnancy (15 to 24 weeks of gestation), and has become expensive as significant license fees are being levied on laboratories running human chorionic gonadotropin analyses using conventional methods (Auxter, S., Clin. Labor News 23: 1-3 (1997)).
Definitive prenatal diagnosis of fetal chromosome abnormalities leading to Down""s syndrome, which affect 1 in 700 live births, typically involves instead culture of amniocytes at midtrimester gestation. The procedure involves the aspiration of a small sample of amniotic fluid (amniocentesis), culturing of the fetal cells contained in the fluid, and determination of the karyotype of these cells and thus the fetus. The major indications for the use of this technique for the detection of chromosome abnormalities are maternal age (usually offered to all mothers over the age of 35 at the time of expected delivery), the presence of a parental chromosome abnormality, or a maternal history of carrying a previous trisomic child or aborted fetus karyotyped to be trisomic. Direct transcervical aspiration of chorionic villi (chorionic villus sampling, or CVS) has also been used for prenatal diagnosis.
Though both procedures have been shown to be relatively safe and reliable, it is generally accepted that they involve some risk, including risk of miscarriage, and, in the case of CVS, also risk of limb hypoplasia in babies born following the procedure. It would be desirable to have other methods for screening pregnant women for Down""s syndrome fetuses, particularly screens that are noninvasive and sensitive. Most Down""s syndrome cases occur in younger pregnant women, those under 35 at the time of expected delivery, or the majority of pregnancies. Less invasive screening tests are needed employing serum or urine samples to identify those at high risk for Down""s syndrome pregnancies, who may not want the risk of amniocentesis or CVS.
Human chorionic gonadotropin (hCG) is a glycoprotein hormone secreted in relatively large quantities by the trophoblast cells of the placenta (Masure, H. R., et al., J. Clin. Endocrinol. Metab. 53: 1014-1020 (1981)). hCG is composed of two dissimilar subunits, xcex1 (92 amino acids and two N-linked oligosaccharides) and xcex2 (145 amino acids and two N-linked and four O-linked oligosaccharides), joined noncovalently, and is detected in the serum and urine of pregnant women and in those with trophoblast disease (hydatidiform mole and choriocarcimoma). Free xcex1- and free xcex2-subunits, and degraded hCG and free xcex2-subunit molecules are also detected in serum and urine samples (Birken, S., et al., Endocrinology 122: 572-583 (1988)). The degraded molecules include nicked hCG and nicked free xcex2-subunit, each cleaved between xcex2-subunit residues 47 and 48 (or less commonly between residues 43 and 44 or 44 and 45), nicked xcex2-subunit missing all or part of the C-terminal sequence (xcex293-145) and, a terminal degradation product comprising two fragments, xcex2-subunit sequences 6-40 and 55-92, held together by disulfide linkages, found primarily in urine samples (FIG. 1). The terminal degradation product has no O-linked oligosaccharides and degraded N-linked oligosaccharide moieties, one or two N-linked pentasaccharides, versus two undecasaccharides on free xcex2-subunit and hCG (FIG. 2A). The terminal degradation product was called xcex2-core fragment (xcex2-core, Blithe, D., et al., Endocrinology 122: 173-180 (1988)), firstly because of its small size (xcx9c9,000 daltons; hCG is 37,000 daltons), and secondly because of its retention of hCGB radioimmunoassay or xcex2-submit core antisera (versus C-terminal or other) immunoreactivity (Birken, et al., and Masure, et al., cited above). Through most of a pregnancy, xcex2-core fragment is the principal hCG xcex2-subunit-related molecule in urine samples.
Serum and urine free xcex2-subunit derive from three sources: direct section by trophoblast cells, slow dissociation of circulating hCG into free xcex1- and xcex2-subunits, and by the nicking of hCG by macrophage or neutrophil enzymes associated with trophoblast tissue, and the more rapid dissociation of nicked hCG in circulation (FIG. 1). Free xcex2-subunit may be nicked by nicking enzymes in the circulation. Urine xcex2-core fragment appears to derive from the degradation of nicked free xcex2-subunit in the kidney.
In the late 1980s, the triple marker test mentioned above was developed to screen for Down syndrome pregnancies. It combined maternal age with serum measurements of hCG, xcex1-fetoprotein, and unconjugated estriol (Bogart, M. H., et al., Prenat. Diagn. 7:623-630 (1987), U.S. Pat. No. 4,874,693 to Bogart, Wald, N. J., et al., Br. J. Obstet. Gynaecol. 95: 334-341 (1988), and Canick, J. A., J. Clin. Immunoassay 13: 30-33 (1990)). More recently, serum-free xcex2-subunit tests and free xcex2-subunit-xcex1-fetoprotein combinations have been introduced as alternative Down syndrome-screening methods (Macri, J. N., el al., Am. J. Obstet. Gynecol. 163: 1248-1253 (1990) and Spencer, K., et al., Ann. Clin. Biochem. 30: 394-401 (1993)). The best serum free xcex2-subunit combination, or the optimal triple marker test, however, detects only 60 to 65 percent of Down""s syndrome cases, with a 5 percent false-positive rate. At these detection and false-positive rates, approximately 80 amniocenteses need to be performed to detect a single case of Down syndrome, and a significant number of Down""s syndrome cases are missed (Cole, L. A., et al., Prenatal Diagnosis 17: 607-614 (1997)). There is a need for improvement in prenatal screening methods.
It is an object of the invention to provide a prenatal screening test for Down""s syndrome pregnancies.
It is another and more specific object of the invention to provide a sensitive, noninvasive test for Down""s syndrome fetuses in pregnant women.
It is a further object of the invention to provide an improvement in the triple marker test that exhibits a decreased false positive rate.
These and other objects are accomplished by the present invention, which provides a novel diagnostic method for screening for the presence or absence of Down""s syndrome in the fetus of a pregnant woman which comprises obtaining a biological test sample from the woman and determining the presence of Down""s syndrome by observation of hyperglycosylated gonadotropin in the sample. This typically involves measuring the concentration of hyperglycosylated gonadotropin, its free xcex2-subunit, its free xcex1-subunit, and/or its xcex2-core fragment in the test sample, and determining the presence of Down""s syndrome by observation that the concentration in the test sample population differs from a normal hyperglycosylated gonadotropin or free xcex1-subunit, free xcex2-subunit, or xcex2-core fragment population and/or is the same as, or similar to, a Down""s syndrome population. In preferred embodiments, the test sample is urine, saliva, plasma or serum, the population comprises hyperglycosylated hCG, xcex2-core fragment, free xcex1-subunit, free xcex2-subunit, and mixtures thereof, and any differences between the properties observed in the normal and Down""s syndrome samples reflect differences observed in the carbohydrate content of the glycopolypeptides and/or glycopeptides.
Carbohydrate compositional or structural analyses, immunoassays, and combinations of these methods are generally employed. In some embodiments, hyperglycosylated gonadotropin is determined directly by assay for at least one hyperglycosylated species, i.e., variant hCG, free xcex2-subunit, free xcex1-subunit, and/or xcex2-core fragment. These screens typically employ a monoclonal, polyclonal, or fusion phage antibody to a hyperglycosylated or carbohydrate-variant hCG species in an ELISA, Western blot, or the like. In another embodiment, elevated levels of monosaccharides are observed in samples positive for Down""s syndrome. Other screens employ lectins that bind the carbohydrate moieties, chromatography, chemical or electrophoresis or isolelectric focussing tests that detect glycosylation variants of hCG. Lectins may also be employed to separate and/or concentrate hCG species having aberrant carbohydrate moieties prior to immunoassay.